Hemostatic compositions

ABSTRACT

The invention relates to a hemostatic composition in powder form comprising collagen of the fibrillar type comprising a content of fibrous collagen and/or fibrillar collagen of at least 70% by weight relative to the total weight of the collagen, and at least one monosaccharide, and optionally, at least one compound selected from coagulation factors and glycosaminoglycans. The invention further relates to a method for preparing such composition, and to a unit comprising such composition and a spraying device.

FIELD OF THE INVENTION

The present invention relates to the field of hemostatic compositions,to the use of specific compounds or compositions as a hemostatic agent,to a method for preparing a hemostatic composition and to a hemostaticmethod.

TECHNICAL BACKGROUND

Wounds, whether external or internal, traumatic or surgical, frequentlylead to bleeding. Such bleeding can be more or less significant.Bleeding is prevented and stopped via a set of physiological phenomenacalled “hemostasis”. Hemostasis helps repair the vascular breach and,generally, ensures the maintenance of vessel and tissue integrity.

When a blood vessel is injured, a natural mechanism comprising variousstages is triggered to stem the flow of blood. First, vasoconstriction,which slows the bleeding, lasts for 15 to 60 seconds and induces acomplex cascade of reactions. A fibrous mesh composed of fibrin formsaround the platelet plug: the final thrombus is formed and is protectedfrom premature dissolution by factor XIII, which stabilizes fibrin.Finally, the fibrin mesh draws tighter (retraction) and the edges of thewound come together: the wound shrinks. Within the stable, cross-linkedfibrin, fibroblasts can then grow and organize into a conjunctive matrixwithin the thrombus and finally close the wound.

No solid fibrin is present in circulating blood; if it were it wouldimmediately obstruct vital vessels. However, fibrin's precursor,fibrinogen, is present. Under the action of thrombin, whose synthesis isactivated by coagulation factors, fibrinogen is transformed intoinsoluble fibrin.

Lastly, several days or weeks after successful healing of the wound, thefibrin cluster is destroyed during fibrinolysis.

In spite of this biochemical phenomenon, it is often necessary, inparticular in the case of wounds that are too large or in the case ofdiffuse bleeding, to “artificially” carry out hemostasis.

There are “mechanical” solutions to help obtain hemostasis, such aspressure, ligature and electrocoagulation, which are used as first-linetreatments. However, these solutions have little or no effectiveness ina certain number of cases, such as oozing capillary hemorrhages,hemorrhages of hypervascularized organs such as the spleen or liver,hemorrhages leading to diffuse bleeding, for example bones, and/or inneurosurgery.

“Chemical” solutions, in particular implemented in certain currenthemostatic products, also exist. The components of said chemicalsolutions are in general either of the “absorbent” or “active” type.

Absorbent hemostatic products, notably comprising polysaccharides suchas regenerated oxidized cellulose or alginates, function mainly bymechanical action and simple absorption. They frequently present aproblem of excessive swelling. If said swelling leads to rapidabsorption of liquid, in particular blood, it can also lead toundesirable pressure when used in a “closed” environment, for example incontact with the dura mater or in urology.

In addition, certain products, notably those comprising plantpolysaccharides such as cellulose or alginates, can further causeinflammatory reactions during their resorption and/or can lead todegradation products not recognized by the host. The consequence of thisis that it is desirable to remove such products so that they do notremain in the body and thus do not produce these adverse effects.

Active hemostatic products, such as products containing thrombin orfibrin, are often blood-derived products. Such products involve risks ofallergies and disease transmission, in particular in the case where thedisease vector would not be inactivated by classically appliedtreatments. In addition, said downstream treatments are generallycomplex and/or costly. Lastly, in general they can require preparationbefore use, which can be a constraint, indeed a nuisance, in terms of anemergency.

Moreover, products containing both fibrin and thrombin base their modeof action on the interaction between the two blood-derived productscomprising the product. The reaction can occasionally take place withoutinteraction with the blood, in which case the products are said tofloat. In other words, the product is pushed away by the blood whichcontinues to flow, possibly causing the product to become diluted or tocoagulate and form a gel on top of the blood, a situation in which theflow of blood is not blocked. Hemostasis can thus not be achieved.

The products available today on the market can be in the form of:

-   -   sponges or matrices: this physical form facilitates handling but        can be problematic during use in complex areas or in        laparoscopic operations,    -   fibers: this form can be easily positioned in the area to be        treated, if the area is accessible, and can reduce the quantity        of products to be implanted; on the other hand, it is strongly        contraindicated on vessels, notably to avoid passage of fibers        into circulation, because such a situation presents risks of        embolism as well as risks of sticking to instruments,    -   solidifying liquids, such as biological adhesives: such        compositions are often comprised of at least two products, which        are mixed together during preparation in order to react and to        polymerize. Said products generally must be prepared just before        use, notably in the operating room, which makes their        preparation tedious and requires their use to be anticipated,    -   sprays: adhesives can be in spray form requiring the use of a        particular device for connecting the syringe to an apparatus        providing the air necessary to propel the product. The dose,        homogeneity of distribution and/or thickness of the layer        deposited are difficult or impossible to regulate.    -   Powder: some powders made out of starch are available on the        market. The hemostatic powder is reached thanks to the        absorption of the blood by the powder. The starch does not have        any biological properties towards the blood. The density and        granulometry (size grading) of this powder is very low and the        powder cannot be sprayed without forming a cloud which is not        suitable for laparoscopic applications. For instance, the powder        described in WO 2005/072700 is not suitable for a homogeneous        and precise spray on the wound, especially in laparoscopy.

The compositions of the prior art are generally provided in the form ofsponges, fibers or viscous liquids, such as fibrin adhesives, that canbe sprayed. However, no powder suitable to be sprayed is available atthe present time.

The present invention thus aims at obtaining a composition that solvesall or some of the problems mentioned above, and in particular acomposition that is satisfactory with regard to safety, effectiveness,ease-of-use and/or cost. In particular, the product or composition canmeet regulatory approval according to regulations in force in eachcountry.

More precisely, the invention aims at obtaining a composition withsatisfactory absorption capacity, good hemostatic capacity, minimumadverse effects (or no adverse effects), good capacity to anchor on theedge of the wound and the possibility to be sprayed, satisfactorypenetration in the blood flow when sprayed and/or limited swelling.

In particular, the composition aims to be able to be administered inmost surgical procedures, such as laparotomies, laparoscopies,coelioscopies and robotic surgical techniques, and/or can be sprayed.The composition and metabolites thereof can preferentially havesatisfactory harmlessness, have improved safety, cause a weak or noimmunogenic response, not be carcinogenic and/or be resorbable. Inparticular, resorption is compatible with the phenomena of hemostasisand healing in order to be able to be left in place after the procedure.

The composition can further aim to be provided in a form that can besprayed.

In addition to solving all or some of the problems mentioned above, thepresent invention can also aim at obtaining a composition:

-   -   making it possible to obtain a product, in particular a dry,        sprayable powder comprising all the components useful for        hemostatic action, in particular during its storage, in other        words, a product that can be stored in a form that is        ready-to-use and that does not require mixing just before use,        and/or    -   whose principal component or whose components do not absorb so        much blood that there is a risk of undesirable pressure, and/or    -   whose components simultaneously have hemostatic, mechanical and        biochemical, or biological activity, and/or    -   which can be used immediately without preparation, without        handling of the hemostatic product, and which can be, for        example, of use in laparoscopy as well as in laparotomy.

SUMMARY OF THE INVENTION

According to a first aspect, the invention thus relates to a hemostaticcomposition comprising, or constituted of:

-   -   collagen comprising a fibrous and/or fibrillar collagen content        of at least 50% by weight relative to the total weight of the        collagen, that is a collagen of the fibrillar type.    -   at least one monosaccharide and    -   optionally, at least one compound selected from:        -   coagulation factors, in particular thrombin, which can in            particular come from blood or be obtained by            biotechnological methods, and        -   glycosaminoglycans, in particular chondroitin sulfate,            dermatan sulfate, hyaluronic acid and mixtures thereof.

Preferably, there is proposed a hemostatic composition in powder formcomprising collagen of the fibrillar type comprising a content offibrous collagen and/or fibrillar collagen of at least 70% by weightrelative to the total weight of the collagen, and at least onemonosaccharide.

The present invention can in particular make it possible to obtain acomposition with particularly good hemostatic properties, notably thanksto the characteristics of size and/or density of the particles of saidcomposition and to the components thereof, while producing limitedswelling.

According to one variant, the composition is free of components obtaineddirectly or indirectly from blood. When thrombin is present, forexample, it can be blood extracted or recombinant thrombin. Quiteparticularly, the composition is constituted of compounds that arerapidly degraded in the body and/or are biocompatible, in other words,that do not induce an undesirable response.

Preferred but non limiting aspect of the composition, taken alone or incombination, are the following:

-   -   the composition comprises a collagen content ranging from 80% to        90% by weight relative to the total weight of the composition.    -   the composition comprises a monosaccharide content ranging from        1% to 12.5% by weight relative to the total weight of the        composition.    -   the composition comprises a collagen/monosaccharide weight ratio        ranging from 10 to 50, and preferably of 19.    -   the collagen comprises a content of fibrous collagen and/or        fibrillar collagen ranging from 85% to 95% by weight relative to        the total weight of the collagen.    -   the composition further comprises a glycosaminoglycan content        ranging from 2% to 25% by weight relative to the total weight of        the composition.    -   the composition further comprises a collagen/total carbohydrate        compounds weight ratio ranging from 2 to 40, wherein the weight        of the total carbohydrate compounds is the sum of the weight of        the monosaccharide(s) and the weight of the        glycosaminoglycan(s).    -   the composition further comprises coagulation factor, in        particular thrombin, in an amount ranging from 0.01 IU/mg to 20        IU/mg of the composition.    -   the composition has a tapped density greater than 0.4 g/mL.    -   the composition comprises at least 50% by weight of particles        whose size is between 200 μm and 400 μm.    -   the composition of any of claims 1 to 10, comprising:        -   a collagen of in an amount of 86.36% by weight relative to            the total weight of the composition,        -   glucose, in an amount of 4.54% by weight relative to the            total weight of the composition,        -   chondroitin sulfate, in an amount of 9.09% by weight            relative to the total weight of the composition.    -   the composition further comprises thrombin, in an amount of 0.2        IU/mg to 2 IU/mg of the composition.

According to a further aspect, the invention relates to a method forpreparing a hemostatic composition comprising at least the followingsteps:

-   -   a) formation of an aqueous suspension comprising a collagen of        fibrillar type having a content of fibrous collagen and/or        fibrillar collagen of at least 70% by weight relative to the        total weight of the collagen, and at least one monosaccharide,        wherein the collagen used in this step is preferably obtained by        basic extraction;    -   b) recovery of the product in the form of precipitate, paste or        gel, notably by centrifugation or decantation,    -   c) drying of the product,    -   d) grinding of the product to the desired granulometry.

Such method may comprise a step e) subsequent to the step d), consistingin adding least one compound selected from:

-   -   coagulation factors, notably thrombin, and    -   glycosaminoglycans, in particular chondroitin sulfate, dermatan        sulfate, hyaluronic acid and mixtures thereof

The invention further relates to a kit comprising the above describedcomposition and a spraying device adapted to contain and propel suchcomposition.

BRIEF DESCRIPTION OF THE DRAWINGS

Other characteristics and advantages of the invention will become clearfrom the following description which is only given for illustrativepurposes and is in no way limitative and should be read with referenceto the attached drawings on which FIG. 1 is an example of a result of anelectrophoresis as described in example 13.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, absent a statement to the contrary, weightpercentages are given relative to the total dry weight of thecomposition.

In the context of the present invention, “total dry weight of thecomposition” refers to the total weight of the composition free ofsolvent, in particular water, and thus the total weight relative to theanhydrous product.

In addition, the weights of the components and the resulting percentagescan correspond to the anhydrous weight of these components, in otherwords, to the weight of the component not including the water which itcould contain. This can also be applied to the percentages obtained.

The composition can comprise a collagen content greater than or equal to70% by weight relative to the total weight of the composition, inparticular greater than or equal to 75% by weight, in particular greaterthan or equal to 77% by weight, indeed greater than or equal to 80% byweight.

In addition, the composition can comprise a collagen content less thanor equal to 99% by weight relative to the total weight of thecomposition, in particular less than or equal to 96% by weight, inparticular less than or equal to 93% by weight, indeed less than orequal to 90% by weight.

Thus, the composition can comprise a collagen content ranging from 70%to 99% by weight relative to the total weight of the composition, inparticular ranging from 75% to 96% by weight, in particular ranging from77% to 93% by weight, indeed ranging from 80% to 90% by weight.Preferably, the content of collagen is around 86% by weight of the totalweight of the composition.

Collagen is the main structure protein in mammals. Collagen consists oftropocollagen (TC) molecules that have lengths around 280-300 nm anddiameters of around 1.5 nm.

The term “fibrous collagen” refers to collagen in the form of fiber,corresponding to an assembly of fibrils. Fibers generally have adiameter ranging from 1 μm to 10 μm. The term “fibrillar collagen”refers to collagen in the form of fibrils. More precisely, fibrilsgenerally have a diameter of 10 nm to 1 μm. Thus, fibrils are formedfrom staggered arrays of tropocollagen molecules, and these fibrils maybe arranged to form collagen fibers. Fibrous and/or fibrillar collagenis generally not soluble, whereas non-fibrillar collagen is highlysoluble.

The definition of fibrous collagen and fibrillar collagen can be inparticular that given by Markus Buehler in “Nature designs toughcollagen: explaining the nanostructure of collagen fibrils,” in PNAS,Aug. 15, 2006, vol. 103, no. 33, pp. 12285-12290.

More than 28 different collagens have been discovered and are classifiedin 3 main categories: collagens of the fibrillar type, collagens of thenon-fibrillar type, and FACIT collagens.

Collagens of the fibrillar type are collagens that mostly comprisefibrillar and/or fibrous collagens and hardly any non-fibrillarcollagens (for example collagen of type I). Similarly, collagens of thenon-fibrillar type are collagens that mostly comprise non-fibrillarcollagens. Some collagens of the non-fibrillar type may consist only innon-fibrillar collagens (for example collagen of type IV or V).

The industrial extraction and purification of collagen generallyconsists in the destructuration of the initial tissues to 1) removeevery or the majority of contaminant proteins and 2) to obtain therequested structuration level depending on the final use of the product.Collagen extraction is generally performed in acid or basic conditionsthat allow the solubilisation of monomolecular soluble collagen which isnot fibrillar. The final collagen naturally contains a mix offibrillar/fibrous collagen and non-fibrillar collagen. The proportionbetween fibrillar/fibrous collagen and non-fibrillar collagen depends onthe tissue chosen for the extraction and the extraction process.

The final product is different than a collagen that has been obtained byan artifical mix of only fibrillar collagen and only non-fibrillarcollagen. In the article entitled “Extraction of collagen fromconnective tissue by neutral salt solutions” (Proceedings of theNATIONAL ACADEMY OF SCIENCES Volume 41 Number I Jan. 15, 1955 by JérômeGross, John H. Highberger and Francis O. Schmitt), are shown thedifferences between fibrillar and non-fibrillar collagens obtained aftera specific extraction process which leads—as described previously—to amix of those two collagens.

In the present composition, the collagen is of the fibrillar type, andcomprises fibrous and/or fibrillar collagen in an amount of at least 60%by weight, in particular at least 70% by weight, in particular at least75% by weight, indeed at least 80% by weight relative to the totalweight of the collagen.

More particularly, the collagen comprises at least 85%, in particular atleast 90%, in particular at least 95%, indeed at least 98% by weight offibrous and/or fibrillar collagen relative to the total weight of thecollagen in the composition.

Preferably the composition comprises a content of fibrous and/orfibrillar collagen ranging from 85% to 95% by weight relative to thetotal weight of the collagen in the composition, and most preferablyfrom 85% to 90% by weight.

This means that in the preferred embodiment, the composition thuscomprises a content of non-fibrillar collagen ranging from 5% to 15% byweight relative to the total weight of the collagen in the composition,and most preferably from 10% to 15% by weight.

It is very advantageous to have a composition with such proportion offibrous and/or fibrillar collagen relative to the non-fibrillarcollagen, in particular for use as a hemostatic powder. Indeed, thefibrous and/or fibrillar collagen should be present in a sufficientamount to perform the hemostasis, and the non-fibrillar collagen shouldalso be in a sufficient amount for the cohesion of the product and notin a too large amount to avoid excess of swelling.

The collagen can be selected among type I collagens or type I and IIIcollagens. The collagen can be extracted from various source tissues, inparticular skin and/or tendons, from all species, more particularlyporcine, bovine or equine species.

The collagen can mostly be made of fibrous collagen of porcine originextracted from skin and/or tendons. In the case of collagen extractedfrom tendons, the extraction can be such as described in internationalapplication WO 2010/125086.

The aforesaid collagen, in particular fibrous and/or fibrillar collagen,can come from acid or basic extraction. According to a particularembodiment, said collagen comes from basic extraction. According to aparticular embodiment, the collagen can be such as described in patentapplication FR2944706.

Preferably, the collagen comes from a basic extraction that enablesmaximizing the content of fibrous and/or fibrillar collagen in theextracted collagen. Further, such basic extraction can be optimized forcontrolling the proportion of the fibrillar/fibrous collagen and thenon-fibrillar collagen within the extracted collagen. Unlike the acidicextraction, the basic extraction allows the hydrolysis of proteoglycans.This action leads to the destructuration of the tissue and theseparation of the fibers without modification of their shape. In acidicconditions, the swelling of the inner collagen molecules in the fibersleads to their partial destructuration during the process with therelease of greater amount of non-fibrillar soluble collagen.

The collagen can be cross-linked, notably by classic modes ofcross-linking such as thermal dehydration, the use of bridging agents,for example formaldehyde and/or glutaraldehyde; by oxidizedpolysaccharides, for example according to the method described ininternational application WO 2010/125086; and/or by oxidizedamylopectins or glycogen.

The composition comprises at least one monosaccharide, alone or inmixture with other monosaccharides. Said monosaccharides can be selectedfrom ribose, sucrose, fructose, glucose and mixtures thereof. Themonosaccharide present in the composition of the invention, alone or inmixture with monosaccharides, is in particular glucose.

The composition can comprise a monosaccharide content ranging from 1% to12.5% by weight relative to the total weight of the composition, inparticular ranging from 1.5% to 10% by weight, in particular rangingfrom 2% to 8% by weight, and quite particularly ranging from 2.5% to7.5% by weight. Most preferably, the monosaccharide content is around 5%by weight relative to the total weight of the composition.

The composition can comprise a collagen/monosaccharide weight ratioranging from 5 to 100, in particular from 7 to 65, more particularlyfrom 10 to 50, and still more particularly from 11 to 40. Mostpreferably, the composition comprises a collagen/monosaccharide weightratio of around 19.

The monosaccharide, notably ribose, sucrose, fructose, glucose andmixtures thereof, and in particular glucose, can notably make itpossible to obtain particles comprising mainly fibrous and/or fibrillarcollagen and monosaccharides with the desired characteristics, notablyof size and density. Incorporation of monosaccharide in the mixture ofcollagen further allows reduction of the electrical charges within thecomposition, which enables forming a powder adapted to be placed withincontainer such as tubes, blower, spraying dispenser.

Quite particularly, the presence of monosaccharide can make it easierand/or cheaper to obtain particles of a desired density and/or size, inparticular in terms of improving the hemostatic properties of a powderof the composition.

Grounding collagen fibers without any additives leads to the reductionof the size of the fibers and lowers the density of the powder. Further,the final preparation contains important amount of electrical chargesthat prevent the manipulation of the final product. It is thus proposedto add monosaccharide before grounding of the collagen, which leads to ahardening of the preparation to mix allowing a rapid grounding(limitation of denaturation), thus enabling preparation of a powder withreduced electrical charges (suitable for placing the powder intocontainers, such as spray dispenser) and a final density suitable forspraying the composition.

Unlike what could have been expected such adjunction of monosaccharidehas no effect on the final activity of the product. In particular, itdoes not modify the bioactivity of the final product. The monosaccharidehas no hemostatic effects.

Further, such adjunction of the monosaccharide does not make it behavingas a foaming agent as it is the case in WO 01/97873. In WO 01/97873, theheating of the diluted solution leads to the formation of gelatin. Highconcentration of gelatin can be made to obtain very concentratedsolution, but the final product contains gelatin and not collagen.Gelatin is known to be less hemostatic than collagen as plateletaggregation needs the presence of collagen fibrils and structure of thenative collagen which are absent in gelatin.

According to one embodiment, the composition comprises, indeed consistsof, particles comprising, indeed consisting of, collagen andmonosaccharide, notably selected from ribose, sucrose, fructose, glucoseand mixtures thereof, in particular glucose.

The composition can comprise at least one, in particular one,coagulation factor. Said coagulation factors are well known to thosepersons skilled in the art. Preferentially, the coagulation factor isthrombin.

Said coagulation factor, in particular thrombin, can come from animalsources (extracted from animal tissues and fluids) or from recombinantsources (produced by cultures of genetically modified cells). Thecoagulation factor may for example be thrombin extracted from humantissues.

When a coagulation factor, in particular thrombin, is present, itscontent can be less than 0.1% by weight relative to the total weight ofthe composition.

In the case of thrombin, international units (IU) are generally used.Thus, the composition can comprise a thrombin content ranging from 0.01IU/mg to 20 IU/mg of the composition, in particular from 0.05 IU/mg to10 IU/mg, in particular from 0.1 IU/mg to 5 IU/mg, indeed from 0.2 IU/mgto 2 IU/mg. Most preferably the content of thrombin—if any—is around0.83 I U/mg of the composition.

In addition to the monosaccharide, the composition can comprise at leastone other carbohydrate compound, which can be a glycosaminoglycan. Suchcarbohydrate compound may be part of the composition, with or without acoagulation factor such as thrombin.

Said glycosaminoglycan can be selected from chondroitin sulfates,dermatan sulfate, hyaluronic acid and mixtures thereof, in particularchondroitin sulfates.

Glycosaminoglycans can make it possible to improve the speed at whichblood is absorbed by the powder. More particularly, glycosaminoglycanscan accelerate contact between the blood and the hemostatic products, inparticular collagen and thrombin.

The composition can comprise a glycosaminoglycan content ranging from 2%to 25% by weight relative to the total weight of the composition, inparticular ranging from 3% to 20% by weight, in particular ranging from4% to 15% by weight, quite particularly ranging from 5% to 12.5% byweight. Most preferably the content of glycosaminoglycan—if any—isaround 9% by weight of the total weight of the composition.

The composition can comprise a collagen/glycosaminoglycan weight ratioranging from 2.5 to 50, in particular from 3.5 to 35, more particularlyfrom 5 to 25, and still more particularly from 6.5 to 20.

According to one embodiment, the composition comprises at least one, inparticular one, monosaccharide and at least one, in particular one,glycosaminoglycan, notably such as defined above, and in particular inthe amounts defined above.

The carbohydrate compounds are quite particularly monosaccharides andglycosaminoglycans.

The composition can comprise a carbohydrate content ranging from 2% to25% by weight relative to the total weight of the composition, inparticular ranging from 5% to 23% by weight, in particular ranging from7% to 21% by weight, quite particularly ranging from 10% to 18% byweight.

The composition can comprise a collagen/carbohydrate compound weightratio ranging from 2 to 40, in particular from 2.5 to 30, moreparticularly from 3 to 20, and still more particularly from 3.5 to 15.

The expression “total weight of carbohydrate compounds” refers to thesum of the weight of the monosaccharides defined above and the weight ofthe other carbohydrate compounds mentioned above.

According to one embodiment, the composition comprises, indeed consistsof:

-   -   collagen comprising mainly a fibrous and/or fibrillar collagen        content of at least 50% by weight relative to the total weight        of the collagen, and    -   at least one, in particular one, monosaccharide.

Quite particularly, the composition comprises, indeed consists of:

-   -   collagen, notably in an amount ranging from 70% to 99% by weight        relative to the total weight of the composition, in particular        ranging from 75% to 96% by weight, in particular ranging from        77% to 93% by weight, indeed ranging from 80% to 90% by weight,        wherein said collagen comprises a fibrous and/or fibrillar        collagen content of at least 50% by weight relative to the total        weight of the collagen, and    -   at least one monosaccharide, in particular glucose, in an amount        ranging from 1% to 12.5% by weight relative to the total weight        of the composition, notably ranging from 1.5% to 10% by weight,        in particular ranging from 2% to 8% by weight, and quite        particularly ranging from 2.5% to 7.5% by weight.

According to another embodiment, the composition comprises, indeedconsists of:

-   -   collagen comprising mainly a fibrous and/or fibrillar collagen        content of at least 50% by weight relative to the total weight        of the collagen,    -   at least one, in particular one, monosaccharide,    -   at least one, in particular one, coagulation factor.

Quite particularly, the composition comprises, indeed consists of:

-   -   collagen, notably in an amount ranging from 70% to 99% by weight        relative to the total weight of the composition, in particular        ranging from 75% to 96% by weight, in particular ranging from        77% to 93% by weight, indeed ranging from 80% to 90% by weight,        wherein said collagen content comprises a fibrous and/or        fibrillar collagen content of at least 50% by weight relative to        the total weight of the collagen,    -   at least one monosaccharide, in particular glucose, in an amount        ranging from 1% to 12.5% by weight relative to the total weight        of the composition, in particular ranging from 1.5% to 10% by        weight, in particular ranging from 2% to 8% by weight, and quite        particularly ranging from 2.5% to 7.5% by weight, and    -   at least one, in particular one, coagulation factor, in        particular thrombin, in an amount ranging from 0.01 IU/mg to 20        IU/mg of the composition, in particular from 0.05 IU/mg to 10        IU/mg, in particular from 0.1 IU/mg to 5 IU/mg, indeed from 0.2        IU/mg to 2 IU/mg.

According to another embodiment, the composition comprises, indeedconsists of:

-   -   collagen comprising mainly a fibrous and/or fibrillar collagen        content of at least 50% by weight relative to the total weight        of the collagen,    -   at least one, in particular one, monosaccharide, and    -   at least one, in particular one, glycosaminoglycan.

Quite particularly, the composition comprises, indeed consists of:

-   -   collagen, notably in an amount ranging from 70% to 99% by weight        relative to the total weight of the composition, in particular        ranging from 75% to 96% by weight, in particular ranging from        77% to 93% by weight, indeed ranging from 80% to 90% by weight,        wherein said collagen content comprises a fibrous and/or        fibrillar collagen content of at least 50% by weight relative to        the total weight of the collagen,    -   at least one monosaccharide, in particular glucose, in an amount        ranging from 1% to 10% by weight relative to the total weight of        the composition, in particular ranging from 1% to 12.5% by        weight, in particular ranging from 1.5% to 10% by weight, in        particular ranging from 2% to 8% by weight, and quite        particularly ranging from 2.5% to 7.5% by weight, and    -   at least one glycosaminoglycan, in particular chondroitin        sulfate, in an amount ranging from 2% to 25% by weight relative        to the total weight of the composition, in particular ranging        from 3% to 20% by weight, in particular ranging from 4% to 15%        by weight, quite particularly ranging from 5% to 12.5% by        weight.

According to still another embodiment, the composition comprises, indeedconsists of:

-   -   collagen comprising a fibrous and/or fibrillar collagen content        of at least 50% by weight relative to the total weight of the        collagen,    -   at least one, in particular one, monosaccharide,    -   at least one, in particular one, coagulation factor, and    -   at least one, in particular one, glycosaminoglycan.

Quite particularly, the composition comprises, indeed consists of:

-   -   collagen, notably in an amount ranging from 70% to 99% by        weight, in particular ranging from 75% to 96% by weight, in        particular ranging from 77% to 93% by weight, indeed ranging        from 80% to 90% by weight relative to the total weight, in        particular to the dry weight, of the composition, wherein said        collagen comprises a fibrous and/or fibrillar collagen content        of at least 50% by weight relative to the total weight of the        collagen,    -   at least one monosaccharide, in particular glucose, in an amount        ranging from 1% to 10% by weight relative to the total weight of        the composition, notably ranging from 1% to 12.5% by weight,        notably ranging from 1.5% to 10% by weight, in particular        ranging from 2% to 8% by weight, and quite particularly ranging        from 2.5% to 7.5% by weight,    -   at least one coagulation factor, in particular thrombin, in an        amount ranging from 0.01 IU/mg to 20 IU/mg of the composition,        in particular from 0.05 IU/mg to 10 IU/mg, in particular from        0.1 IU/mg to 5 IU/mg, indeed from 0.2 IU/mg to 2 IU/mg, and    -   at least one glycosaminoglycan, in particular chondroitin        sulfate, in an amount ranging from 2% to 25% by weight relative        to the total weight of the composition, notably ranging from 3%        to 20% by weight, in particular ranging from 4% to 15% by        weight, quite particularly ranging from 5% to 12.5% by weight.

According to a quite particular embodiment, the composition comprises,indeed consists of:

-   -   collagen of the fibrillar type, mostly comprising fibrous and/or        fibrillar collagen, said collagen of the fibrillar type being        for example obtained by extraction in basic medium, and being in        an amount of around 85% by weight relative to the total weight        of the composition,    -   glucose, in an amount of around 4.9% by weight relative to the        total weight of the composition,    -   thrombin, in an amount of 0.2 IU/mg to 2 IU/mg of the        composition, and    -   chondroitin sulfate, in an amount of around 10% by weight        relative to the total weight of the composition.

According to another particular embodiment, the composition comprises,indeed consists of:

-   -   collagen of the fibrillar type, mostly comprising fibrous and/or        fibrillar collagen, said collagen of the fibrillar type being        for example obtained by extraction in basic medium, and being in        an amount of around 85% by weight relative to the total weight        of the composition,    -   glucose, in an amount of 5% by weight relative to the total        weight of the composition,    -   and chondroitin sulfate, in an amount of 10% by weight relative        to the total weight of the composition.

In the context of the present invention, the expression “an amount ofaround X %” refers to a variation of plus or minus 20%, in other words,an amount of around 10% means from 8% to 12%, in particular a variationof plus or minus 10%, indeed plus or minus 5%.

When the thrombin is added, the powder of thrombin is mixed with thepowder of the homogeneous molecular mixture of collagen/monosaccharidealready prepared. When chondroitin sulfate are added, the thrombin isfirst added to the chondroitin sulfate powder and this mix is added tothe previous mixture of collagen/monosaccharide (already ground intopowder). The thrombin is not stabilized neither by carbohydrate norcollagen. The thrombin is never in contact with a solution of themonosaccharide (contrary to WO 98/57678) which prevents any denaturationof the protein and a rehydration of the powder leading to animpossibility to dry it again properly.

The composition can be provided in several forms of solid powders, inparticular a sprayable powder. The composition of the invention ispreferably a sprayable powder.

The composition in powder form can in particular comprise, or consistof:

-   -   particles comprising, or consisting of, collagen of the        fibrillar type and at least one monosaccharide, in particular        glucose, wherein in particular said particles have a size,        granulometry and/or density such as defined in the present        description, and    -   optionally, particles comprising, or consisting of, at least one        glycosaminoglycan, in particular chondroitin sulfates, and/or at        least one coagulation factor, in particular thrombin, wherein in        particular said particles have a size, granulometry and/or        density such as defined in the present description.

The present composition in powder form, quite particularly a sprayablepowder, can advantageously have a tapped density greater than 0.4 g/L,quite particularly greater than or equal to 0.41 g/L. Indeed, when usinga powder as an hemostatic agent, the density has to be enough to preventany dilution and repulsion of the product by the blood flow. Providing apowder composition with a density greater than 0.4 g/L prevents thepowder to be flushed away by the blood.

When applied manually, the surgeons should compress the powder with agauze without removing the product at the end of the compression.

When the powder composition is intended to be sprayed upon a bleedingwound, it is preferable that such composition has a much higher densityto allow the powder granules to reach a certain kinetic energy whenejected from the sprayer. Indeed, powder with a low density cannot beperfectly moved by air to reach the wound without the formation of acloud, which is not compatible to the precise administration needed inmost cases. Having a high density also enables the sprayer to impart thepowder a sufficient kinetic energy for a good impact of the powder ontothe wound. Thus, the powder composition has preferably a tapped densityhigher than 0.4 g/mL. Even more preferably, the powder composition has atapped density lower than or equal to 0.6 g/mL.

A way to obtain a powder composition with such density features consistsin grounding a cake of dry collagen, such cake being a hard and densepreparation of collagen. The required density cannot be reached bygrounding a lyophilized preparation of collagen, because the finalparticles would be flat and thus not sprayable, with an importantelectrostatic charge. For instance, the product as prepared in WO01/97873 cannot be reduced to a powder having such a density and suchsprayable features.

The powder described in WO 2005/072700 has a very low density, and thispowder cannot thus be sprayed to reach a targeted organ especially inlaparoscopy. In particular, it will not reach the wound with therequired impact preventing repulsion from the blood flow.

The present composition, in particular in powder form, quiteparticularly sprayable, can preferentially have a tapped density higherthan 0.4 g/L, in particular higher than 0.41 g/L.

Even more preferably, the tapped density of the composition powder ishigher than 0.4 g/mL, and most preferably lower than or equal to 0.6g/mL.

The bulk density is generally greater than or equal to 0.3 g/L, quiteparticularly greater than or equal to 0.31 g/L.

The bulk density is advantageously higher than 0.3 g/L, quiteparticularly higher than 0.31 g/L.

These densities are measured according to the protocols described in theexamples.

The composition of the invention advantageously has a hemostaticcapacity ranging from 1 second to 600 seconds, in particular from 1second to 200 seconds, more particularly from 1 second to 60 seconds,indeed from 1 second to 40 seconds. Hemostatic capacity is measuredaccording to the protocol presented in the examples.

The composition of the invention advantageously comprises at least 50%by weight of particles whose size is between 200 μm and 400 μm.

The particles constituting the powder advantageously have a meangranulometry ranging from 10 μm to 500 μm, in particular from 50 μm to400 μm.

Advantageously, at least 90% by weight, in particular 100% by weight, ofthe particles constituting said powder can pass through a screen whosemesh is 500 μm, in particular 400 μm.

At least 90% by weight, and in particular at least 95% by weight, of theparticles constituting said powder can be retained by a screen whosemesh is 10 μm, notably 20 μm, indeed 30 μm, indeed 50 μm.

This repartition has been chosen to allow the powder to be sprayed. Withparticles size too small, the powder is too pulverulent to reachproperly the target and with particle size too high the powder cannot besprayed

The majority compound of the composition, in particular collagen, canhave a swelling ratio at 20 minutes ranging from 0.7 to 1, as measuredaccording to the protocol described in the examples (see example 4).

The composition, in particular when said composition comprises at leastone glycosaminoglycan, and in particular when said composition compriseschondroitin sulfate, can have a swelling ratio at 20 minutes rangingfrom 5 to 15, in particular from 8 to 12, and quite particularly from 10to 12, as measured according to the protocol described in the examples(see example 5).

According to a preferred embodiment, the composition is provided in theform of a sprayable dry powder.

The composition in powder form comprises in particular:

-   -   particles comprising collagen and a monosaccharide, and    -   optionally, at least glycosaminoglycan and/or thrombin.

The composition can comprise:

-   -   particles comprising collagen, a monosaccharide and optionally        at least one glycosaminoglycan and/or coagulation factor,    -   particles comprising collagen, a monosaccharide and optionally a        coagulation factor and optionally glycosaminoglycan particles,    -   particles comprising collagen and a monosaccharide and particles        comprising at least one glycosaminoglycan and/or coagulation        factor.

In the context of the present invention, the expression “dry powder”means that the composition comprises a limited content of solvent, inparticular water. Said limited content can be less than 20% by weight,in particular less than 15% by weight, in particular less than 10%, andquite particularly less than 5% by weight relative to the total weightof the composition.

Said dry form can be obtained by simple evaporation of the solvent used,by dehydration by organic solvents.

The composition in dry powder form can particularly be administered byspraying.

In particular, the invention relates to a kit comprising:

-   -   a composition according to the invention, and    -   a spraying device.

The composition is such as defined according to the present description.Those persons skilled in the art know of various spraying devices forspraying pharmaceutical compositions in powder form.

In particular, the device can comprise a reservoir of pressurized gas,wherein said pressure is released by activating means of activation, inparticular by a trigger.

The device can comprise:

-   -   means of activation,    -   a powder reservoir, in particular comprising a powder according        to the invention,    -   a gas reservoir, connected to the powder reservoir,    -   a sprayer connected to the powder reservoir,    -   a valve A coupled with the means of activation, wherein said        valve A is closed when the means of activation are at rest and        thus blocking the flow of gas between the gas reservoir and the        powder reservoir, and wherein said valve is open when the means        of activation are activated in order to deliver an injection of        gas to spray at least part of the powder.

The gas reservoir can be coupled with means of supplying pressurizedgas, for example a system of compressed gas or a tank of compressed gas,in particular in order to make the system autonomous.

The device can comprise, between the gas reservoir and the means ofsupply, a valve B, wherein said valve B is coupled with the means ofactivation.

The valve B is by default in the open position, except when the means ofactivation are activated, in which case it is closed.

Thus, when the valve A is open, the valve B is closed, and vice versa.This enables the delivery of a predetermined quantity of pressurized gasto deliver a dose of powder while preventing contaminants fromcontacting the interior of the device.

When the device is not coupled with means of supplying gas the valve Bis preferably closed. This can help limit the risks of contamination ofthe interior of the device.

Quite particularly, the powder reservoir is in the form of a cylindercomprising several doses of powder. Said cylinder can be coupled with apropeller activated by the flow of gas enabling the powder to be sprayedand thus turning the cylinder and changing the dose of powder afteractivation of the means of activation.

The doses present in the cylinder can be sealed in order to be sterile,in particular with a component that tears open when the flow of gascrosses the aforesaid dose. This makes it possible to preserve the dosesin a sterile environment.

The volume of a dose can be around 0.2 cm³ to 1 cm³, in particulararound 0.3 cm³.

The gas used can be air, carbon dioxide or any inert gas.

In particular, the device is such as described in European patentEP11163820.1 and U.S. provisional application No. 61/479,805 both filedApr. 27, 2011, and incorporated by reference.

The present invention also relates to a method for preparing ahemostatic composition, notably such as defined above, comprising atleast the following steps:

-   -   a) formation of an aqueous suspension comprising, indeed        consisting of, collagen of the fibrillar type—mainly comprising        fibrous and/or fibrillar collagen—and a monosaccharide, such as        glucose,    -   b) recovery of the product in the form of precipitate, paste or        gel, notably by centrifugation or decantation,    -   c) drying of the product, for example by evaporation.    -   d) grinding of the product to the desired granulometry, in        particular by a hammer mill, and    -   e) optionally, adding thrombin and/or chondroitin sulfates,        notably in solid form, in particular in powder form.

The formation in step a) of an aqueous suspension comprising, thefibrous/fibrillar collagen and a monosaccharide leads to a homogeneousrepartition of the monosaccharide around the collagen molecules.Further, the close contact between the molecular species of collagen andthe monosaccharide leads, after deshydration, to a hard cake suitablefor obtaining—by grounding—a powder with the required high density. Onthe contrary, mixing a collagen powder and a glucose powder does notlead to an homogeneous and sprayable powder, in particular because ofthe density and electrical charges.

In step a) the collagen can be present at a concentration ranging from30 g/L to 150 g/L.

The monosaccharide can be added to the suspension or to the homogeneouscollagen paste in an amount such as defined in the description, and moreparticularly from around 2% to 5% by weight relative to the weight ofthe collagen.

In step a) the monosaccharide can be present at a concentration rangingfrom 0.3 g/L to 10 g/L.

The aqueous suspension of collagen of step a) can be acid, and inparticular comprise an acid such as hydrochloric acid. Said acid can bepresent at a concentration ranging from 0.01 M to 0.5 M, and inparticular from 0.02 M to 0.1 M, indeed around 0.05 M. Said suspensioncan be in the form of homogeneous paste.

Step b) can comprise the pouring of the suspension into a mold.

Step c) is performed so as to obtain a cake as thick as possible(superior the final granulometry wanted), with a very high density andas less air bubbles as possible (less than 5%) inside the cake.

Step d) can be followed by a step of screening of the powder, notably inorder to obtain the desired granulometry.

According to a preferred embodiment, step a) consists in forming amixture comprising 95% by weight of collagen of the fibrillar type and5% by weight of glucose. After having dried (step b)) and ground (stepc)) this mixture, chondroitin sulfate is added in a content of 10% byweight of the total weight of the mixture, such that the finalcomposition comprises:

-   -   collagen: 86.36% by weight relative to the total weight of the        composition;    -   glucose: 4.54% by weight relative to the total weight of the        composition;    -   chondroitin sulfate: 9.09% by weight relative to the total        weight of the composition;

When thrombin is also added, it represents a final content lower than0.01% by weight relative to the total weight of the composition. In theabove mixture, thrombin may be in an amount of 0.083 IU/mg of thecomposition.

According to another embodiment, the invention also relates to thepowders obtainable by the method of the invention.

For all the aforesaid powder products, it is quite obviously possible toapply a more or less thorough grinding to obtain a powder of variablegranulometry according to the type of grinding and the duration thereof.

According to another of its aspects, the present invention relates tothe use of a composition such as defined above as a hemostatic agent.

According to still another of its aspects, the invention relates to apharmaceutical composition or drug comprising, indeed consisting of, acomposition such as defined above, in particular as a hemostatic agent.

According to another of its aspects, the invention relates to the use ofa composition such as defined above for preparing a drug orpharmaceutical composition, in particular a hemostatic drug orpharmaceutical composition.

According to another of its aspects, the present invention relates to ahemostatic method comprising the depositing and/or the spraying of acomposition such as defined above on a hemorrhaging part of an animal'sbody. In particular, the composition can be used in surgical procedures,in particular laparotomies, laparoscopies, coelioscopies and roboticprocedures.

The invention also relates to a hemostatic method comprising a step ofspraying a composition of the invention.

According to another of its aspects, the present invention relates tothe use of a composition such as defined above as a biological adhesive.

According to still another of its aspects, the present invention relatesto the use of a composition such as defined above as a cicatrizing agentfor internal and external wounds. The expression “cicatrizing agent”refers to a product that makes it possible to obtain a clinicallysatisfactory cicatrization of the tissues with which it is in contact.

EXAMPLES Example 1 Protocol for Measuring Density

The product to be tested (600 mg), in particular in powder form, isintroduced into a 5 mL graduated cylinder. The height of the product ismeasured and the corresponding volume is noted. The measurement isrepeated three times. The product is then packed three times by tappingthe tube 100 times on a hard surface. The height of the product and thecorresponding volume are measured again. The densities of the productbefore and after packing, called bulk density and tapped density,respectively, are thus obtained.

Example 2 Protocol for Measuring Hemostatic Capacity In Vitro

Citrated (around 0.1 M) human blood is maintained at 37° C. in a waterbath throughout the measurement. The product to be tested (10 mg) isdeposited in a 5 mL polypropylene tube with a snap-on cap, and thencitrated fresh blood (2 mL) is added. CaCl₂ is then added so that thefinal CaCl₂ concentration in the blood is 15 mM, and then the test tubeis closed. The contents are then around mixed by vigorous inversions (10times) and then the test tube is plunged into the water bath; the testtube is returned to the vertical position every 10 seconds. The timerequired to form a clot is noted and corresponds to hemostatic capacity.

Example 3 Protocol for Measuring Particle Size

A known quantity of product, notably of powder, is sifted through 50 μm,100 μm, 200 μm, 300 μm and 400 μm screens for 2 minutes (per screen).The fractions from each screen are weighed. The proportion of eachgranulometry range is determined.

Example 4 Protocol for Measuring the Swelling of the Majority Compoundof the Composition

The compound, in particular collagen (40 mg to 70 mg) is weighed in aweighing boat. In another weighing boat, 6 mL of 0.15 M NaCl isdeposited. The compound to be tested is deposited on 0.15 M NaCl (t=0).After 20 minutes, excess liquid is removed and the swollen product isweighed. The swelling ratio corresponds to the quantity of waterabsorbed in relation to the weight of the dry starting product.

Example 5 Protocol for Measuring the Swelling of the Composition

A 15 mL flask is weighed (m₀ in mg) and then X mg of powder of the drycomposition is added (m_(0+x) in mg). A 0.15 M aqueous NaCl solution (2mL) is added and the composition is left to swell for 20 minutes; theflask is then centrifuged at 1,000 rpm.

Excess NaCl is removed with a Pasteur pipette and droplets areeliminated by turning over the flask on filter paper; the flask is thenweighed with the wet powder (m₁ in mg).

The swelling ratio is calculated as follows: ((m₁−m₀)/(m_(0+x)−m₀)).

Example 6 Preparation of Collagen of the Fibrillar Type by BasicExtraction

Pieces of pig dermis (30 kg), defatted with acetone, are left to swellfor 3 hours in 100 kg of 0.05 M NaOH solution. The dermises are finelycut up by a cutting mill and the paste obtained is diluted with 50liters of 0.05 M NaOH. The mixture is then sieved under pressure througha 1 mm screen. The paste obtained is then brought to pH 6-7.5 with HCland the precipitate obtained is collected by centrifugation orfiltration through a 1 mm screen.

The retentate is dehydrated with acetone according to methods known tothose persons skilled in the art. This dehydrated retentate thusconsists in collagen of the fibrillar type, with a large content offibrillar/fibrous collagen relative to the non-fibrillar collagen.Generally, such extracted collagen comprises from 85% to 95% by weightof fibrillar/fibrous collagen relative to the total weight of thecollagen, and from 5% to 15% by weight of non-fibrillar collagenrelative to the total weight of the collagen.

Example 7 Preparation of a Hemostatic Powder #1

30 g of collagen of the fibrillar type as prepared in Example 6 is addedto 1 L of a 0.02 M aqueous HCl solution and the mixture is then stirredfor 5 hours. Next, to the homogeneous paste obtained, powdered fructoseis added in an amount of 2% (0.6 g) by weight relative to the weight ofthe collagen.

The mixture is homogenized for 1 hour and then poured out anddehydrated. After drying, the dry product is ground at a rate of 25g/min using a Fitzpatrick hammer mill at 7,000 rpm under controlledheating. The product is then screened by mechanical sifting to eliminateparticles whose size is larger than 400 μm.

Dermatan sulfate is then added to the powder in an amount of 2% byweight relative to the dry matter of the powder (0.612 g).

The mixture is then homogenized using a ball mill, lyophilized thrombinis added to the mixture in an amount of 15 IU/mg of powder, and finallythe mixture is homogenized using a ball mill.

Example 8 Preparation of a Hemostatic Powder #2

7.5 kg of collagen of the fibrillar type as prepared in Example 6 isadded to 50 L of a 0.05 M aqueous HCl solution and the mixture is thenstirred for 16 hours. Next, to the homogeneous paste obtained, powderedfructose is added in an amount of 5% (375 g) by weight relative to theweight of the collagen.

The mixture is homogenized for 3 hours and then distributed onto platesand dehydrated. After drying, the dry product is ground by fraction at arate of 5 g/min using a hammer mill at 12,000 rpm under controlledheating. The product is then screened by mechanical sifting to eliminateparticles whose size is larger than 400 μm and those smaller than 50 μm.

Granulometry is measured in order to verify that the distribution issuch that 60% of the sample by weight has a granulometry greater than200 μm.

Purified chondroitin sulfates are then added to the powder in an amountof 20% by weight relative to the dry matter of the powder (1.575 kg).The mixture is homogenized using a ball mill.

Finally, lyophilized thrombin is added to the mixture in an amount of 10IU/mg of powder. As before, the mixture is homogenized using a ballmill.

Example 9 Preparation of a Hemostatic Powder #3

1000 g of collagen of the fibrillar type as prepared in Example 6 isadded to 60 mL of a 0.02 M aqueous HCl solution and the mixture is thenstirred for 5 hours. Next, to the homogeneous paste obtained, powderedglucose is added in an amount of 5% (50 g) by weight relative to theweight of the collagen.

The mixture is homogenized for 1 hour and then poured out anddehydrated. After drying, the dry product is ground at a rate of 25g/min using a Fitzpatrick hammer mill at 7,000 rpm under controlledheating. The product is then screened by mechanical sifting to eliminateparticles whose size is larger than 400 μm and smaller than 50 μm.

Chondroitin sulfate is then added to the powder in an amount of 10% byweight relative to the dry matter of the powder (105 g). The mixture isthen homogenized using a ball mill.

Such powder composition has a tapped density of around 0.408 g/mL.

Example 10 Preparation of a Hemostatic Powder #4

500 g of collagen of the fibrillar type as prepared in Example 6 isadded to 30 mL of a 0.02 M aqueous HCl solution and the mixture is thenstirred for 5 hours. Next, to the homogeneous paste obtained, powderedglucose is added in an amount of 5% (25 g) by weight relative to theweight of the collagen.

The mixture is homogenized for 1 hour and then poured out anddehydrated. After drying, the dry product is ground at a rate of 25g/min using a Fitzpatrick hammer mill at 7,000 rpm under controlledheating. The product is then screened by mechanical sifting to eliminateparticles whose size is larger than 400 μm and smaller than 50 μm.

Chondroitin sulfate mixed with a thrombin powder is then added to thepowder in an amount of 10% by weight relative to the dry matter of thepowder (52.5 g). The thrombin is added to the mixture in a final amountof 0.85 U/mg. The mixture is then homogenized using a ball mill.

Such powder composition has a tapped density of around 0.425 g/mL.

Example 11 Performance of Hemostatic Collagen Based Powder #3

The collagen based powder #3 as prepared in example 9 containingcollagen, glucose, chondroitin sulfate, without thrombin, was testedduring in vivo experimentations.

The protocol of the experimentation was the following:

-   -   Wounds were created on the left paramedian liver lobe surface,        of a size about 10*10*1-3 mm³.    -   A product under test was then applied during 30 seconds.        Sufficient product was applied to cover the wound; the test        material was held at the wound site by gentle compression using        the gauze.    -   Then a bleeding grading was made to assess the percentage of        haemostasis reached. If clinically acceptable haemostasis was        reached, then the experimentation was stop. Otherwise, another        compression-grading run was done, up to 5 runs of compressions.        The product under experimentation was renewed at each run.

The experimentation has been performed with the following products: agauze without any haemostatic composition (called “Control”), a gauzecovered by the haemostatic powder #3 (called “GHP #3”), the haemostaticgauze “Avitene” from Davol (called “Avitene”, composed of collagenfibers), the haemostatic gauze “Surgicel” from Ethicon (called“Surgicel”, composed of oxidized cellulosis).

The results are summarized in the following table n° 1, showing thepercentage of haemostasis reached for each product:

TABLE n° 1 Run 1 Run 2 Run 3 Run 4 Run 5 Control 18.2% 36.4% 36.4%63.6%  63.6%  GHP #3 81.8%  100%  100% 100% 100% Avitene 85.7% 85.7%71.4% 100% 100% Surgicel 71.4%  100%  100% 100% 100%

This experimentation shows that the proposed haemostatic powder is veryefficient very quickly, as the haemostasis is full after only two runs,with an efficiency higher than 80% only after the first run.

For “Avitene”, the decrease of the percentage of haemostasis reachedbetween Run2 and Run3 is due to the fact that the wound has bled again.

Example 12 Performance of Hemostatic Collagen Based Powder #4

The collagen based powder #4 as prepared in example 10 containingcollagen, glucose, chondroitin sulfate, and thrombin, was tested duringin vivo experimentations.

The protocol of the experimentation was the following:

-   -   Animal model: Heparinized swines    -   Abdominal surgery:        -   Midline laparoscopy; Liver exposition; With scalpel,            tangential liver wounds of about 10*10*1-3 mm³;        -   A product under test was applied so that wound is well            covered; Compression during 2 min;        -   Bleeding grading (scale from 0 to 4); If success, than stop            at Run 1; Otherwise, new runs of compression and bleeding            scoring; Up to 5 runs allowed.    -   Iliac crest surgery:        -   Dorsal lateral skin and muscle incisions to expose the iliac            crests; Corticotomy with spongy bone exposure (defects of            about 12*12*3 mm³);        -   Product under test was applied so that wound is well            covered; Compression during 2 min;        -   Initial bleeding scoring and further bleeding grading (scale            from 0 to 4); If success, than stop at Run 1; Otherwise, new            runs of compression and bleeding scoring; Up to 5 runs            allowed.

The experimentation has been performed with the following products: agauze without any haemostatic composition (called “Control”), a gauzecovered by the haemostatic powder #4 (called “GHP #4”), the haemostaticgauze “Floseal” from Baxter (called “Floseal”, composed of gelatinmatrix gel+thrombin), the haemostatic gauze “Surgicel” from Ethicon(called “Surgicel”, composed of oxidized cellulosis).

-   -   The results were the following: Abdominal surgery (n=8 to 10        repetitions)—results given after 10 min of compression (=5 runs        of 2 min each)        -   Control: 10/10 failure        -   GHP #4: 11/11 success        -   Floseal: 9/9 success        -   Surgicel: 8/10 success; 2/10 failure    -   Orthopeadic (n=4 repetitions)—results given after 10 min of        compression (=5 runs of 2 min each)        -   Control: 1/4 success; 3/4 failure        -   GHP #4: 4/4 success        -   Floseal: 3/4 success; 1/4: failure        -   Surgicel: 4/4 success

This experimentation shows that the proposed haemostatic powder is 100%efficient for any kind of wounds, contrary to the products of the marketwhich are more wound specific.

Example 13 Collagen Characterization→Presence of Soluble Collagen in theCollagen, Determination of the Ratio Between Fibrillar/Fibrous Collagenand Non-Fibrillar Collagen

The goal of the experimentation is to determine the proportion offibrillar/fibrous collagen and non-fibrillar collagen in a collagen(extracted collagen or collagen ground into powder). Such proportion canbe determined by studying the proportion of insoluble (corresponding tothe fibrillar/fibrous collagen) and soluble collagen (corresponding tothe non-fibrillar collagen) in the collagen.

The experimentation consists in solubilizing about 2.5 g of the collagenunder test in 166 mL of water at pH 13 during 16 hours. The solution isthen centrifuged (10 000 rpm during 10 minutes). The supernatant(corresponding to the non-fibrillar collagen) and the residue(corresponding to the fibrous/fibrillar collagen) are then split. Theresidue is directly dried with successive acetone baths and under acontrolled air flow. The pH of the supernatant is adjusted at pH 3 withacetic acid and chlorhydric acid at 6M. The solid collagen from thesupernatant is obtained by adding NaCl 0.6M, and by performing acentrifugation. It is then dried with successive acetone baths and undera controlled air flow.

The collagen weights from the residue (Mresidue) and from thesupernatant (Msupernatant) are calculated, and the formulaMresidue/(Mresidues+Msupernatant)×100 gives the percentage of fibrouscollagen on total amount of collagen.

In the invention, the ratio Mresidue/(MResidues+Msupernatant) must besuperior to 80% both for the collagen used to prepare the powder and forthe final collagen powder. Preferentially the ratio is superior to 85%.

For example, the above experimentation made of three batches of collagenprepared as in example 6 gives very similar ratios of 92.67%, 94.60% and91.51% respectively. After having ground the collagen of these threebatches, the ratio remains very similar as it is of 91.63%, 88.02%, and88.69% respectively.

Another way to show the presence of both fibrous/fibrillar collagen andsoluble collagen is to perform a SDS page electrophoresis.

FIG. 1 illustrates such electrophoresis, with sample S1 corresponding tothe supernatant of a first batch (made from collagen extracted as inexample 6), sample S2 corresponding to the residue of this first batch,and sample S3 corresponding to the supernatant of a second batch (alsomade from collagen extracted as in example 6), sample S4 correspondingto the residue of this second batch.

The results show that for the collagen from the residue, a larger amountof fiber cannot migrate through the acrylamide gel and are stained atthe stop of the gel. The preparation of the sample does not allow thesplit of each chain from the collagen. Therefore, alpha chains arepresent in a very low amount. The collagen from the supernatant is ableto entirely migrate in the gel, there are no fiber blocked at the top,chains from the collagen are properly split during the electrophoresisprocess.

BIBLIOGRAPHIC DATA

-   WO 2005/072700-   “Nature designs tough collagen: explaining the nanostructure of    collagen fibrils,” by Markus Buehler (PNAS, Aug. 15, 2006, vol. 103,    no. 33, pp. 12285-12290)-   “Extraction of collagen from connective tissue by neutral salt    solutions” by Jérôme Gross, John H. Highberger and Francis O.    Schmitt (Proceedings of the NATIONAL ACADEMY OF SCIENCES Volume 41    Number I Jan. 15, 1955)-   WO 2010/125086-   FR2944706-   WO 01/97873

1.-17. (canceled)
 18. A hemostatic composition in powder formcomprising: collagen of the fibrillar type comprising a content offibrous collagen and/or fibrillar collagen of at least 70% by weightrelative to the total weight of the collagen, and at least onemonosaccharide.
 19. The composition of claim 18, wherein saidcomposition comprises a collagen content ranging from 80% to 90% byweight relative to the total weight of the composition.
 20. Thecomposition of claim 18, wherein said composition comprises amonosaccharide content ranging from 1% to 12.5% by weight relative tothe total weight of the composition.
 21. The composition of claim 18,wherein said composition comprises a collagen/monosaccharide weightratio ranging from 10 to 50, and preferably of
 19. 22. The compositionof claim 18, wherein the collagen comprises a content of fibrouscollagen and/or fibrillar collagen ranging from 85% to 95% by weightrelative to the total weight of the collagen.
 23. The composition ofclaim 18, wherein said composition further comprises a glycosaminoglycancontent ranging from 2% to 25% by weight relative to the total weight ofthe composition.
 24. The composition of claim 18, wherein saidcomposition further comprises a collagen/total carbohydrate compoundsweight ratio ranging from 2 to 40, wherein the weight of the totalcarbohydrate compounds is the sum of the weight of the monosaccharide(s)and the weight of the glycosaminoglycan(s).
 25. The composition of claim18, wherein said composition further comprises coagulation factor, inparticular thrombin, in an amount ranging from 0.01 IU/mg to 20 IU/mg ofthe composition.
 26. The composition of claim 18, wherein saidcomposition has a tapped density greater than 0.4 g/mL.
 27. Thecomposition of claim 18, wherein said composition comprises at least 50%by weight of particles whose size is between 200 μm and 400 μm.
 28. Ahemostatic composition in powder form comprising: collagen of thefibrillar type comprising a content of fibrous collagen and/or fibrillarcollagen of at least 70% by weight relative to the total weight of thecollagen, wherein said collagen is in an amount of 86.36% by weightrelative to the total weight of the composition, glucose, in an amountof 4.54% by weight relative to the total weight of the composition,chondroitin sulfate, in an amount of 9.09% by weight relative to thetotal weight of the composition.
 29. The composition of claim 28,further comprising thrombin, in an amount of 0.2 IU/mg to 2 IU/mg of thecomposition.
 30. A method for preparing a hemostatic compositioncomprising at least the following steps: a) formation of an aqueoussuspension comprising a collagen of fibrillar type having a content offibrous collagen and/or fibrillar collagen of at least 70% by weightrelative to the total weight of the collagen, and at least onemonosaccharide, b) recovery of the product in the form of precipitate,paste or gel, notably by centrifugation or decantation, c) drying of theproduct, d) grinding of the product to the desired granulometry.
 31. Themethod of claim 30, wherein the collagen used in step a) is obtained bybasic extraction.
 32. The method of claim 30, further comprising a stepe) subsequent to the step d), consisting in adding least one compoundselected from: coagulation factors, notably thrombin, andglycosaminoglycans, in particular chondroitin sulfate, dermatan sulfate,hyaluronic acid and mixtures thereof.
 33. A kit comprising: a sprayingdevice, and a hemostatic composition in powder form comprising: collagenof the fibrillar type having a content of fibrous collagen and/orfibrillar collagen of at least 70% by weight relative to the totalweight of the collagen, and at least one monosaccharide.
 34. A kitcomprising: a spraying device, and a hemostatic composition in powderform comprising: collagen of the fibrillar type having a content offibrous collagen and/or fibrillar collagen of at least 70% by weightrelative to the total weight of the collagen, wherein said collagen isin an amount of 86.36% by weight relative to the total weight of thecomposition, glucose, in an amount of 4.54% by weight relative to thetotal weight of the composition, chondroitin sulfate, in an amount of9.09% by weight relative to the total weight of the composition.
 35. Ahemostatic composition for use as a hemostatic agent, being in powderform and comprising: collagen of the fibrillar type having a content offibrous collagen and/or fibrillar collagen of at least 70% by weightrelative to the total weight of the collagen, and at least onemonosaccharide.
 36. A hemostatic composition for use as a hemostaticagent, being in powder form and comprising: collagen of the fibrillartype having a content of fibrous collagen and/or fibrillar collagen ofat least 70% by weight relative to the total weight of the collagen,wherein said collagen is in an amount of 86.36% by weight relative tothe total weight of the composition, glucose, in an amount of 4.54% byweight relative to the total weight of the composition, chondroitinsulfate, in an amount of 9.09% by weight relative to the total weight ofthe composition.